The long term goal of this study is to understand the regulation of cell proliferation in the organ of Corti. Sensory hair cell loss is the major cause of hearing and balance problems affecting the lives of millions of people. In lower vertebrates, hair cells, if lost, can regenerate through the proliferation of supporting cells. In mammals, however, reinitiation of cell proliferation in the damaged sensory epithelia is, at best, very limited, leading to failure in repair of sensory epithelia. In addition, cell proliferation has to be tightly regulated to coordinate growth with morphogenesis during normal development. The understanding of cell proliferation regulation, therefore, represents a central issue in the study of development and regeneration studies of the inner ear. Previously, we have initiated a study to identify regulators for cell proliferation during development of the organ of Corti in mice. We found that p27Kip1, a cell division inhibitor, plays an essential growth inhibitory role during development of the organ of Corti. It may also be an impediment for reinitiating supporting cell proliferation in the mouse organ of Corti following hair cell loss. I propose to study the regulation of cell proliferation in the developing organ of Corti by the ubiquitin (ub)-proteasome pathway, a pathway universally involved in regulating the cell division cycle and, in particular, involved in p27Kip1 regulation in cell lines. The following specific aims are proposed: (1) establish and characterize embryonic cochlear organ culture for the dissection of the involvement of the ub-proteasome pathway in the developing organ of Corti; (2) test the hypothesis that p27Kip1 is regulated by the ub-proteasome pathway by studying the half lives of p27Kip1 during development of the organ of Corti, and in the presence of inhibitors for the ub-proteasome pathway. (3) test the hypothesis that the ub-proteasome pathway regulates cell proliferation in the organ of Corti via the regulation of p27Kip1. Cell proliferation will be examined in the organ of Corti in the presence of proteasome inhibitors. Experiments will then be performed to test whether p27Kip1 is essential and sufficient in mediating cell proliferation control by the ub-proteasome pathway in the organ of Corti; (4) isolate and identify components of the ub-proteasome pathway responsible for the developmental regulation of p27. This specific aim represents a long term direction of the study.